Insulin-dependent diabetes mellitus (IDDM) and celiac disease are two of the most common immune-mediated diseases in humans. In order to develop new treatment for both conditions, we have decided to study the mechanisms underlying their association to the same Major Histocompatibility Complex (MHC) class II molecules, HLA-DQ8. These molecules share with HLA-DQ2, HLA-DR0405 and I-Ag7 a mutation at position 57 of their chain that eliminates a conserved aspartic acid and modifies the charge and shape of the P9 pocket. We have evaluated the functional consequences of this mutation in mouse and human when the bound peptide has no Asp/Glu residue at position 9 (P9). In all instances, this situation leads to the selection of Asp/Glu residues in CDR3 of T cell receptor (TCR). The pairing Asp/Glu in CDR3 with non-Asp/Glu P9 peptide increases TCR/pMHC affinity dramatically and supports T cell expansion. The determination of the first structure of an I-Ag7/T cell receptor complex, coupled with biophysical measurements, revealed that this phenomenon was induced at a distance by non-TCR contact residues through electrostatic forces. In the current proposal we propose to explore further the role that the shift between low and high TCR affinity, related solely on the nature of the P9 residue of the peptide (the P9 switch Model), may have in disease onset and development. To explore this fascinating possibility, we propose 3 specific aims. Aim 1: Evaluation of the role of position 57 of MHC in T cell repertoire selection. We hypothesize that position 57 of MHC directly select T cells of higher affinity when peptides do not carry Asp/Glu at P9. Already shown for exogenous antigens such as gliadin and lysozyme, we will evaluate this mode of recognition for self-antigens such as BDC2.5, GAD65 and insulin in the context of I-Ag7 and HLA-DQ8. Aim 2: Mechanisms supporting the role of position 57 in T cell receptor interactions. We will combine biophysical and structural studies to understand the gain in affinity that occurs when a TCR retaining a Asp/Glu residue in its CDR3 recognizes a peptide with a neutral residue at P9 in the context of I-Ag7 or HLA-DQ8. Aim 3: Using animal models to test the pertinence of the P9 switch model in autoimmune responses. We have circumstantial evidence showing that the T cell recognition of some antigens hinges on the nature of 57 of MHC and the associated peptide. However, we now have to provide a direct link between this unique mode of recognition and the expansion of autoreactive T cells in vivo and show their role in disease. We will address the issue directly by T cell and retrogenic transfer studies in mice carrying I-Ag7 (NOD, C57BL6.g7). In addition, studies in a mutant I-Ag757D mutant mouse will challenge directly our model. We believe that we have identified a unique molecular link between I-Ag7/HLA-DQ8 and T cell recognition and hypothesize that this link supports the expansion of autoreactive T cells in HLA-DQ8- associated diseases. At stake is the possibility to redefine MHC molecules as direct targets for treatment and the development of small molecules or antibodies binding to HLA-DQ molecules to modify their function. PUBLIC HEALTH RELEVANCE: Insulin-dependent diabetes mellitus (IDDM) and celiac disease are two of the most common autoimmune diseases in humans. To develop new therapeutic approaches we are trying to understand the mechanisms of their association with MHC class II molecules such as HLA-DQ8 and propose the first comprehensive model ever to be tested.